Life Science Quarterly

FAQs

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER Winter 2002

 Frequently Asked Questions
 

As a major Life Science supplier, Sigma-Aldrich has spent years helping customers with numerous application and technique inquiries. By using some of the information gained from these customer contacts, we present some of our Frequently Asked Questions (FAQs). These FAQs will hopefully serve as a guide to help identify and resolve some of your concerns.

SYBR Green JumpStart Taq ReadyMix

How is SYBR Green JumpStart Taq ReadyMix different from ABI’s SYBR Green PCR Master Mix?

The formulation is very similar. ABI’s product contains AmpliTaq Gold® (chemically inactivated hot-start polymerase), SYBR Green dye, deoxynucleotides and optimized buffer. A ROX reference dye solution is also included. Sigma’s product is designed to perform comparable to the ABI product, using JumpStart Taq for antibody-inactivated hot start PCR. A proprietary reference dye solution is provided with Sigma’s SYBR Green JumpStart Taq ReadyMix. The solution may be used as a reference for baseline fluorescence, using the same instrument settings as for ROX.

Will this reagent work on all commonly available instruments?

The SYBR Green JumpStart Taq ReadyMix was designed for use on the ABI 7700 instrument. The reagent is expected to also work on the Bio-Rad iCycler, the Stratagene MX4000 and the Cephid Smart Cycler.

SYBR Green JumpStart Taq ReadyMix is not expected to work with the Roche LightCycler. The LightCycler uses a capillary system for thermocycling, and the Roche reagents contain an inert carrier protein to reduce adhesion to the capillary surfaces.

Will this reagent work with TaqMan ® probes or molecular beacons (labeled primers used for qPCR)?

No, the chemistry is different. TaqMan probes and molecular beacons are designed to have complementary sequences to specific DNA templates and contain their own dye moieties. We have not run any trials, but we expect there will be background interference of the SYBR Green dye with the dyes on the probes or beacons. In addition, TaqMan probes and molecular beacons are usually used for multiplex simultaneous PCR. Two or more TaqMan Probes/molecular beacons can be designed with different dyes for different DNA templates in a single solution. When PCR is performed, each dye has a different fluorescent emission wavelength, and amplification of each template can be measured separately from other templates present.

TaqMan probe and molecular beacons allow the customer to target their reaction to a specific template and are suitable for multiplex PCR. However, the primers are significantly more expensive than nonlabeled oligonucleotides. SYBR Green dye is non-specific but is a much cheaper alternative to dye-labeled probes.

I always get CT s in the range of X to Y. What CTs will I get using the Sigma reagent?

The CTs (threshold cycles) with the Sigma reagent will be in the same range, if not a tighter range, than the ABI and Roche reagents and our initial studies show they are superior to Qiagen. Since the JumpStart enzyme is more sensitive and specific than the competitors the CTs will be more reproducible and in a tighter range.

I am using TaqMan probes, molecular beacons, or another labeled probe technology. Can I use SYBR Green JumpStart Taq ReadyMix?

Yes. In this situation, we suggest using the SYBR Green mix as a less expensive reagent to qualify the primer selection prior to making the TaqMan probes or molecular beacons. The manufacturers of qPCR instruments recommend such a qualification prior to running the actual experiments, especially if you will be multiplexing.

What else does Sigma-Aldrich have to offer for quantitative/real-time PCR?

Sigma-Aldrich has a ReadyMix for use with TaqMan probes or molecular beacons. It is Product Code P 2893 , JumpStart Taq ReadyMix for Quantitative PCR. This readymix is comparable to the SYBR Green JumpStart Taq ReadyMix without the addition of the SYBR Green dye for detection of amplification product. The product is listed on the Sigma-Aldrich website, and the technical bulletin is available there.

For advice and information on our products, contact Sigma Technical Services at 1-800-325-5832 or by email at techserv@sial.com

   
 
   
 

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